Project Summary Craniosynostosis is a birth defect defined as the premature fusion of the suture(s) of the skull occurring in 1:1800- 2500 births. The Centers for Disease Control and Prevention, National Birth Defects Study has published data suggesting that ?environmental? exposures including maternal thyroid diseases, use of selective serotonin reuptake inhibitors (SSRIs) in pregnant mothers, and maternal nicotine exposure may exacerbate incidence and or severity of craniofacial anomalies including craniosynostosis. A proposed mechanism of craniosynostosis is the disruption of the balance of proliferation and differentiation of the osteogenic precursors in the perisutural area leading to bone overgrowth within cranial sutures. Recently, undifferentiated cells identified uniquely as Gli1+ or by traditional markers Cd44+ Sca1+ Cd45- Cd34- have been identified in craniofacial bones and sutures. Further, ablation of these undifferentiated cells has been correlated with fusion of the sutures (craniosynostosis). For these reasons, we hypothesize that in utero pharmacological exposures will deplete these multilineage cells, resulting in alterations in craniofacial growth and development. In order to test this hypothesis, we propose to answer whether exposure to pharmacological teratogens in utero deplete defined stem cell populations resulting in altered suture morphology by exposing pregnant wild-type mice to levothyroxine, citalopram, and nicotine and measuring the resultant pups via x-ray cephalometry and a histological assessment of the coronal and posterior interfrontal sutures. In order to investigate depletion of multilineage cells as a mechanism for craniosynostosis, we plan to investigate if pharmacological exposures deplete Cd44+ Sca1+ Cd45- Cd34- cell populations in vitro. We will assess depletion of the cell population of interest in heterogeneous suture derived cells treated with clinically relevant dosages of levothyroxine, citalopram and nicotine. To identify the mechanism of depletion we will sort heterogeneous suture cell populations for the Cd44+ Sca1+ Cd45- Cd34- phenotype, then treat with the pharmacological agents and assess proliferation, differentiation, apoptosis, autophagy, and senescence. Investigating these newly defined cells and their relationship to suture maintenance will provide insight into future manipulation of these cells for therapeutic benefit as a means of decreasing the need for neurosurgical intervention in cases of craniosynostosis.